CN114045331A - Primers for multiple PCR identification of Bungarus parvus medicinal materials, standard decoction and traditional Chinese medicine formula granules, application and identification method thereof - Google Patents
Primers for multiple PCR identification of Bungarus parvus medicinal materials, standard decoction and traditional Chinese medicine formula granules, application and identification method thereof Download PDFInfo
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Abstract
The invention discloses a primer for multiple PCR identification of Bungarus Parvus medicinal materials, standard decoction and traditional Chinese medicine formula granules, which comprises a first primer pair, a second primer pair and a third primer pair; wherein the sequence of the upstream primer of the first primer pair is shown as SEQ ID NO: 1, and the sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification; the sequence of the upstream primer of the second primer pair is shown as SEQ ID NO: 3, the sequence of the downstream primer is shown as SEQ ID NO: 4 is shown in the specification; the sequence of the upstream primer of the third primer pair is shown as SEQ ID NO: 5, the sequence of the downstream primer is shown as SEQ ID NO: and 6. The invention also discloses application of the primer and an identification method based on the primer. The method can effectively identify the bungarus parvus, the red-chain snake and the golden-ring snake at the same time.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine identification, in particular to a primer for multiple PCR identification of Bungarus Parvus medicinal materials, standard decoction and traditional Chinese medicine formula granules, and an application and an identification method thereof.
Background
There are about two hundred species of snake in China, and 20 or more species of snake are common snake, including Agkistrodon Agkistrodon acutus, Zaocys dhumnades, Bungarus multicinctus, Bungarus fasciatus, Ptyas muccosus, Viper Dabois, Naja Naja, Lycoden ruzonatus, Elaphe carinata, Ptyas korros, Gloydius brachuus, Elaphe taeniura, Elaphe moellendorffi, etc., most of which have no clinical efficacy. Because of the similar shapes of various snakes, the medicinal materials are difficult to identify, particularly, after internal organs are cut off during processing, drying and blackening are carried out, the pattern characteristics and the color on the skin almost disappear and are difficult to identify, and particularly, after the snake is prepared into standard decoction through water extraction and further processed into formula particles, the effective identification is more difficult to carry out after the medicinal material properties are lost.
Some studies have indicated that the PCR method (polymerase chain reaction) can be used to identify Agkistrodon acutus. However, the standard decoction and the traditional Chinese medicine formula granules often contain a certain proportion of auxiliary materials, so that the existing PCR method is difficult to apply. In addition, in the preparation process of the standard decoction and the traditional Chinese medicine formula granules, heating extraction is often needed, so that DNA is degraded, DNA fragments of more than 200bp are difficult to retain, and the detection precision of the existing method is low. In addition, the existing PCR method can only realize the identification of a single snake species, and has low efficiency.
Disclosure of Invention
The invention aims to solve the technical problem of providing a primer for multiple PCR identification of Bungarus Parvus medicinal materials, standard decoction and traditional Chinese medicine formula granules, which can effectively identify Bungarus Parvus, Elaphe carinata and Bungarus fasciatus simultaneously.
The technical problem to be solved by the present invention is to provide an application of the above primer.
The technical problem to be solved by the invention is to provide an identification method based on the primer.
In order to solve the technical problems, the invention provides a primer for multiple PCR identification of Bungarus Parvus medicinal materials, standard decoction and traditional Chinese medicine formula granules, which comprises a first primer pair, a second primer pair and a third primer pair;
wherein the sequence of the upstream primer of the first primer pair is shown as SEQ ID NO: 1, and the sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification;
the sequence of the upstream primer of the second primer pair is shown as SEQ ID NO: 3, the sequence of the downstream primer is shown as SEQ ID NO: 4 is shown in the specification;
the sequence of the upstream primer of the third primer pair is shown as SEQ ID NO: 5, the sequence of the downstream primer is shown as SEQ ID NO: and 6.
Correspondingly, the invention also discloses the application of the primer in (1) or (2):
(1) identifying whether the sample to be detected is Bungarus Parvus, Elaphe carinata and Bungarus fasciatus;
(2) preparing a kit for identifying bungarus parvus, Elaphe carinata and/or Bungarus fasciatus;
as an improvement of the technical scheme, the bungarus parvus is a medicinal material, a standard decoction or a Chinese medicinal formula granule;
the red-chain snake is medicinal material, standard decoction or Chinese medicinal formula granule;
the Bungarus fasciatus is prepared from medicinal materials, standard decoction or Chinese medicinal formula granules.
Correspondingly, the invention also discloses the application of the primer in (1) or (2):
(1) identifying whether the sample to be detected contains bungarus parvus, red-chain snake and/or bungarus multicinctus;
(2) preparing a kit for identifying whether the sample to be detected contains bungarus parvus, red-chain snake and/or bungarus multicinctus.
Correspondingly, the invention also discloses a kit which is characterized by comprising the primer.
Correspondingly, the invention also discloses a PCR identification method for the multiple site specificity of the Bungarus Parvus medicinal material, the standard decoction and the traditional Chinese medicine formula granules based on the PCR identification method, which comprises the following steps:
extracting the genome DNA of a sample to be detected;
performing PCR amplification by using the genomic DNA as a template and the primers for multiple PCR identification of the Bungarus Parvus medicinal material, the standard decoction and the traditional Chinese medicine formula particles, wherein if the amplification product contains a DNA band of 160-170bp, the sample to be detected contains the Bungarus Parvus medicinal material, the Bungarus Parvus standard decoction and/or the Bungarus Parvus traditional Chinese medicine formula particles;
if the amplification product contains a DNA band of 201-210bp, the sample to be detected contains a red-chain snake medicinal material, a red-chain snake standard decoction and/or red-chain snake traditional Chinese medicine formula particles;
if the amplification product contains 190-200bp DNA band, the sample to be detected contains a Bungarus fasciatus medicinal material, standard Bungarus fasciatus decoction and/or Bungarus fasciatus traditional Chinese medicine formula particles.
As an improvement of the above technical scheme, the method for extracting the genome DNA of the sample to be detected comprises the following steps:
adding CTAB precipitation liquid and proteinase K precipitation into a sample to be detected, and extracting for 2-3 times; adding CTAB extracting solution and beta-mercaptoethanol into the precipitate for extraction, and then adding chloroform-isoamylol for extraction for 2-3 times; and (3) taking the supernatant after extraction, adding isopropanol or isopropanol-sodium acetate for precipitation and extraction, washing and incubating the precipitate, and dissolving the precipitate with water to obtain the genomic DNA of the sample to be detected.
As an improvement of the above technical scheme, the method for extracting the genome DNA of the sample to be detected comprises the following steps:
taking 0.3-0.8 g of a sample to be detected, grinding the sample to be detected into powder, putting the powder into a centrifuge tube, adding 1-1.8 mL of CTAB precipitation solution and 15-25 mu L of proteinase K, uniformly mixing, heating at 50-60 ℃ for 45-65 min, cooling to room temperature, centrifuging, and removing supernatant; adding 800-1000 mu L CTAB precipitation solution and 15-25 mu L proteinase K, uniformly mixing, heating at 50-60 ℃ for 45-65 min, cooling to room temperature, centrifuging, and removing supernatant; adding 800-1000 mu L of CTAB extracting solution and 5-15 mu L of beta-mercaptoethanol into the precipitate, uniformly mixing, heating at 60-70 ℃ for 100-150 min, cooling to room temperature, taking supernatant, adding chloroform-isoamyl alcohol mixed solution with the same volume, shaking, uniformly mixing, centrifuging, taking 700-800 mu L of supernatant, adding chloroform-isoamyl alcohol mixed solution with the same volume, shaking, uniformly mixing, centrifuging, taking 400-500 mu L of supernatant, adding isopropanol or isopropanol-sodium acetate mixed solution with the same volume, standing at-30 to-20 ℃ for 30-60 min, centrifuging, discarding supernatant, washing the precipitate with ethanol for 2-4 times, discarding supernatant, incubating the precipitate at 35-38 ℃ for 20-40 min, volatilizing ethanol, adding 30-50 mu L of sterilized water for dissolving, and obtaining the genomic DNA of the sample to be detected.
As an improvement of the technical scheme, the CTAB precipitation solution comprises CTAB, Tris-HCl, EDTA and water; wherein the concentration of CTAB is 1-3% (w/v), the concentration of Tris-HCl is 80-120 mmol/L, and the concentration of EDTA is 10-30 mmol/L;
the CTAB extracting solution comprises CTAB, Tris-HCl, EDTA, NaCl, PVP40 and water; wherein the concentration of CTAB is 1-3% (w/v), the concentration of Tris-HCl is 80-120 mmol/L, and the concentration of EDTA is 10-30 mmol/L; the concentration of NaCl is 1-3 mol/L, and the concentration of PVP40 is 10-30% (w/v).
As an improvement of the technical scheme, the template, the primers, the PCR premix solution and water are uniformly mixed to obtain a PCR amplification system;
amplifying the PCR amplification system according to a preset amplification program to obtain an amplification product;
carrying out electrophoretic analysis on the amplification product, recording an electrophoretic map of the amplification product, and if the map contains a DNA strip of 160bp, determining that the sample to be detected contains Bungarus Parvus medicinal materials, Bungarus Parvus standard decoction and/or Bungarus Parvus traditional Chinese medicine formula particles;
if the map contains a DNA band of 207bp, the sample to be detected contains a red-chain snake medicinal material, a red-chain snake standard decoction and/or red-chain snake traditional Chinese medicine formula particles;
if the map contains 191bp DNA bands, the sample to be detected contains the Bungarus fasciatus medicinal material, the standard Bungarus fasciatus decoction and/or the Bungarus fasciatus traditional Chinese medicine formula particles.
As an improvement of the technical scheme, the PCR amplification system consists of the following substances:
PCR premix 12.5. mu.L; 0.3. mu.L of each of the forward primer and the reverse primer of the first primer pair, 0.3. mu.L of each of the forward primer and the reverse primer of the second primer pair, 0.3. mu.L of each of the forward primer and the reverse primer of the third primer pair, 1. mu.L of the template, ddH2O 9.7μL。
As an improvement of the technical scheme, the PCR amplification procedure comprises the following steps:
pre-denaturing the amplification system at 92-95 ℃ for 4-6 min, then circulating for 30-40 times under a preset program, and finally extending at 71-75 ℃ for 4-6 min;
wherein the preset program is: the amplification system is denatured at 94-96 ℃ for 28-32 s, annealed at 60-62 ℃ for 26-32 s, and then extended at 70-73 ℃ for 28-35 s.
As an improvement of the technical scheme, the PCR amplification procedure comprises the following steps:
pre-denaturing the amplification system at 95 ℃ for 5min, then circulating for 35 times under a preset program, and finally extending at 72 ℃ for 5 min;
wherein the preset program is: the amplification system was denatured at 95 ℃ for 30s, then annealed at 61 ℃ for 30s, and then extended at 72 ℃ for 30 s.
As an improvement of the above technical solution, the electrophoretic analysis method comprises: and diluting the amplification product, then spotting the diluted amplification product on 1.5-2% agarose gel, performing electrophoresis for 45-60 min under the condition of 115-125V voltage, and performing Gelred color development to obtain an electrophoresis pattern.
The implementation of the invention has the following beneficial effects:
the primer disclosed by the invention can be used for carrying out multi-site specific PCR reaction, so that the bungarus parvus, the red-chain snake and the golden-ring snake can be effectively identified, and the identification efficiency is improved.
2, the primer and the identification method can effectively overcome the problem of auxiliary material interference in the DNA extraction process, and can solve the problem that standard decoction and traditional Chinese medicine formula granules are difficult to identify after losing forms.
Drawings
FIG. 1 is a graph showing the results of the electrophoresis detection of the snake sample in example 3 after the multiplex PCR amplification; wherein M is DNA molecular weight standard, 1-6 is Bungarus Parvus medicinal material, 7-10 is Bungarus Parvus standard decoction (lyophilized powder), 11-12 is Bungarus Parvus traditional Chinese medicine formula granule, 13-18 is Bungarus Elaphus Seu Periploca medicinal material, 19-24 is Bungarus Elaphus standard decoction (lyophilized powder), 25-30 is Bungarus Parvus medicinal material, 31-36 is Bungarus Elaphus standard decoction (lyophilized powder), 37 is Bungarus Parvus Elaphus (2020061807), Bungarus Elaphus (2020081440), and Bungarus Elaphus (2020102001) medicinal material mixture; 38 is prepared from standard decoction (lyophilized powder) of Bungarus Parvus (2020112020), Elaphe carinata (2020101609), and Bungarus fasciatus (2020101618); n is blank control (ddH)2O)。
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
The invention provides a primer for multiple PCR identification of Bungarus Parvus medicinal materials, standard decoction and traditional Chinese medicine formula granules, which comprises a first primer pair, a second primer pair and a third primer pair, wherein the specific sequences of the primers are shown in the following table:
the primers can carry out multiple PCR reactions in the same PCR reaction system, have no interference with each other and are easy to amplify.
Based on the above characteristics, the primers of the present invention can be applied to:
(1) identifying whether the sample to be detected is the medicinal material, the standard decoction or the traditional Chinese medicine formula granule of the Bungarus Parvus; or the crude drugs, standard decoction and traditional Chinese medicine formula granules of the red-chain snake; or medicinal materials, standard decoction and traditional Chinese medicine formula granules of the golden cypress.
(2) Identifying whether the sample to be detected contains bungarus parvus and/or red-chain snake and/or golden-ring snake; for example, it can be used for identifying Chinese medicinal composition (such as rheumatism and pain relieving capsule) containing Bungarus Parvus.
(3) Preparing a kit for identifying bungarus parvus, Elaphe carinata and/or Elaphe carinata.
(4) Preparing a kit for identifying whether the sample to be detected contains bungarus parvus and/or red-chain snake and/or golden-ring snake.
Correspondingly, the invention discloses a kit, which comprises the primer, PCR premix and water. The PCR premix can be 2 XTaq PCR Master Mix II (Tiangen Biochemical technology (Beijing) Co., Ltd., KT201-12) or 2 Xpfu PCR Mix (Tiangen Biochemical technology (Beijing) Co., Ltd., KP201), but is not limited thereto.
The invention also discloses a multiplex PCR identification method for the Bungarus Parvus medicinal materials, the standard decoction and the traditional Chinese medicine formula granules, which comprises the following steps:
firstly, extracting genome DNA of a sample to be detected;
wherein, the sample to be detected is provided in a solid state form, and the standard decoction is provided in a freeze-dried powder form (i.e. freeze-drying the standard decoction to obtain the freeze-dried powder).
Specifically, the extraction method comprises the following steps:
taking a sample to be detected, adding CTAB precipitation solution and proteinase K precipitation, and extracting for 2-3 times to obtain a precipitate;
specifically, taking 0.3-0.8 g of a sample to be detected, grinding the sample to be detected into powder, putting the powder into a centrifuge tube, adding 1-1.8 mL of CTAB precipitation solution and 15-25 mu L of proteinase K, uniformly mixing, heating at 50-60 ℃ for 45-65 min, cooling to room temperature, centrifuging, and removing supernatant; adding 800-1000 mu L CTAB precipitation solution and 15-25 mu L protease K, uniformly mixing, heating at 50-60 ℃ for 45-65 min, cooling to room temperature, centrifuging, and removing supernatant to obtain the finished product.
The CTAB precipitation solution had a composition of 2% (w/v) CTAB, 100mmol/L Tris-HCl (pH 8.0), and 20mmol/L EDTA (pH 8.0).
(II) adding CTAB extracting solution and beta-mercaptoethanol into the precipitate for extraction; then adding chloroform-isoamyl alcohol for extraction for 2-3 times to obtain supernatant after extraction;
specifically, adding 800-1000 μ L of CTAB extracting solution and 5-15 μ L of beta-mercaptoethanol into the precipitate, uniformly mixing, heating at 60-70 ℃ for 100-150 min, cooling to room temperature, taking supernatant, adding chloroform-isoamyl alcohol (24:1) mixed solution with the same volume, shaking, uniformly mixing, centrifuging, taking supernatant 700-800 μ L, adding chloroform-isoamyl alcohol (24:1) mixed solution with the same volume, shaking, uniformly mixing, centrifuging, and taking supernatant 400-500 μ L to obtain the compound.
In the step, after extraction with the CTAB extract and the β -mercaptoethanol is completed, chloroform-isoamyl alcohol may be directly added, or chloroform-isoamyl alcohol may be added to the supernatant obtained after extraction, and chloroform-isoamyl alcohol may be added to the supernatant to improve the extraction accuracy.
Wherein, CTAB extracting solution: 1-3% (w/v) CTAB, 80-120 mmol/L Tris-HCl (pH 8.0), 10-30 mmol/L EDTA (pH 8.0), 1-3 mol/L NaCl, 10-30% (w/v) PVP 40.
And (III) adding isopropanol or isopropanol-sodium acetate into the extracting solution for precipitation and extraction, washing and incubating precipitates, and dissolving the precipitates with water to obtain the genomic DNA of the sample to be detected.
Specifically, adding equal volume of isopropanol or isopropanol-3 mol/L sodium acetate mixed solution into the extracting solution, standing for 30-60 min at-30 to-20 ℃, centrifuging, removing the supernatant, washing the precipitate with ethanol for 2-4 times, removing the supernatant, incubating the precipitate at 35-38 ℃ for 20-40 min, volatilizing the ethanol, and adding 30-50 mu L of sterilized water to dissolve to obtain the genomic DNA of the sample to be detected.
In addition, when the applicant faces the extraction of genome DNA from standard Bungarus parvus decoction and Bungarus parvus traditional Chinese medicine formula particles, the traditional CTAB method, chelating resin method, triton-100 method, SDS method, alkaline lysis method, DNeasy Blood & Tissue Kit method and magnetic bead method are tried, but all insoluble precipitates are obtained. Therefore, the present inventors have obtained the above extraction method by improving a CTAB precipitation solution, a CTAB extraction solution, and an extraction procedure based on the conventional CTAB method. The extraction method retains genome DNA information of standard Bungarus Parvus decoction and Chinese medicinal granule, and can overcome adjuvant interference.
Forming a PCR amplification system, and carrying out amplification to obtain an amplification product;
specifically, uniformly mixing a template, a primer, a PCR premix solution and water to obtain a PCR amplification system;
more specifically, the total volume of the PCR amplification system was 25. mu.L, which included: PCR premix 12.5. mu.L; 0.3. mu.L of each of the forward primer and the reverse primer of the first primer pair, 0.3. mu.L of each of the forward primer and the reverse primer of the second primer pair, 0.3. mu.L of each of the forward primer and the reverse primer of the third primer pair, 1. mu.L of the template, ddH2O 9.7μL。
Specifically, the PCR premix may be 2 XTAQQ PCR Master Mix II (Tiangen Biochemical technology (Beijing) Co., Ltd., KT201-12) or 2 XPfu PCR Mix (Tiangen Biochemical technology (Beijing) Co., Ltd., KP201), but is not limited thereto.
The amplification procedure was: pre-denaturation at 92-95 ℃ for 4-6 min; denaturation at 94-96 ℃ for 28-32 s, annealing at 60-62 ℃ for 26-32 s, extension at 70-73 ℃ for 28-35 s, and circulating for 30-40 times; finally, extending for 4-6 min at 71-75 ℃.
Preferably, the amplification procedure is: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, extension at 72 ℃ for 30s, and cycle for 35 times, and finally extension at 72 ℃ for 5 min.
(3) Analyzing the amplification product;
specifically, the amplification product can be analyzed by electrophoresis or fluorescent staining, but is not limited thereto.
Preferably, the amplification product is analyzed by electrophoresis, which specifically comprises the following steps:
taking an amplification product, diluting the amplification product, spotting the diluted amplification product on 1.5-2% agarose gel, performing electrophoresis for 45-60 min under the condition of 115-125V voltage, and performing Gelred color development to obtain an electrophoresis pattern.
If the map contains 160bp DNA bands, the sample to be detected contains Bungarus Parvus medicinal materials, standard Bungarus Parvus decoction or Bungarus Parvus traditional Chinese medicine formula particles; if the map contains a DNA strip of 207bp, the sample to be detected contains a red-chain snake medicinal material, a red-chain snake standard decoction or red-chain snake traditional Chinese medicine formula particles; if the map contains 191bp DNA bands, the sample to be detected contains the Bungarus fasciatus medicinal material, the standard Bungarus fasciatus decoction or the Bungarus fasciatus traditional Chinese medicine formula particles.
The present invention will be described below with reference to specific examples.
Example 1 primer design
Using Bungarus Parvus and common snake NADH in China pharmacopoeia 2020 edition; cytochrome Oxidase I; 12S rRNA; 16S rRNA; cytochrome b sequence analysis is taken as a basis, a BioEdit software is utilized to carry out homologous comparison on Cytochrome Oxidase I sequences of common snake species in a GeneBank database, specific SNP sites of the bungarus parvus are analyzed after the sequence is collated, a base sequence containing the SNP sites is introduced into a Primer Premier 5 software, and Primer design is carried out.
Sequence comparison shows that the specific site of the bungarus parvus is T and the others are C, after the SNP site is determined, the SNP site is close to the 3' end of the forward Primer, the position of the upstream Primer is moved, the Primer score and the GC content are adjusted, the position of the reverse Primer is further adjusted by means of Primer Premier 5 software, and the optimal combination is determined by the final Primer score and the product band score. In the design process, the damage effect on the DNA base sequence after high-temperature extraction is also considered.
Combining the above factors, the obtained primer combinations are as follows:
JQBHS-F：5’-GGAGCAATCAATTTTATTACAACAT-3’；
JQBHS-R：5’-GATCGGTTAAAAGTATTGTAACTGC-3’；
CLS-F：5’-ACCGTGTACCCGCCACTG-3’；
CLS-R：5’-AAGCATGATAGCAGTAATTAGCACA-3’
JHS-F：5’-CCTGGGTCACTTCTAGGTAGC-3’；
JHS-R：5’-GCTGGTGGGAGTAGTCAGAAAC-3’。
example 2 identification method establishment
(1) DNA extraction and concentration regulation
Taking 0.5g of a dried sample, grinding the dried sample into powder, placing the powder into a 2mL centrifuge tube, adding 1.5mL of CTAB precipitation solution preheated at 56 ℃ and 20 mu L of proteinase K, uniformly mixing, heating in a 56 ℃ water bath for 60min, cooling to room temperature, centrifuging for 5min at 10000r/min, removing supernatant, adding 900 mu L of CTAB precipitation solution and 20 mu L of proteinase K, and operating in the same way. Sequentially adding 900 mu L of CTAB extracting solution and 10 mu L of beta-mercaptoethanol into the centrifugal tube, uniformly mixing, heating in a water bath at 65 ℃ for 120min, centrifuging, and taking supernatant after cooling to room temperature; adding chloroform-isoamyl alcohol (24:1) with the same volume, shaking for 3min, and mixing; centrifuging at 12000r/min at 4 deg.C for 10min, collecting supernatant 750 μ L, adding into new 2mL centrifuge tube, adding equal volume of chloroform-isoamyl alcohol (24:1), shaking for 3min, mixing, centrifuging at 12000r/min at 4 deg.C for 10 min; and taking 450 mu L of the supernatant, putting the supernatant into a 1.5mL centrifuge tube, adding isopropanol with the same volume, and standing for 30-60 min at-20 ℃. Taking out the centrifuge tube, centrifuging at 12000r/min for 5min, discarding supernatant, washing the precipitate with 75% ethanol and anhydrous ethanol twice, discarding supernatant, incubating the precipitate at 37 deg.C for 30min, volatilizing ethanol, and adding 30 μ L sterilized water to dissolve.
Wherein, the CTAB precipitation solution comprises the following components: 2% (w/v) CTAB (limited biotechnology, Shanghai-derived leaf), 100mM Tris-HCl (pH 8.0) (Beijing Solarbio), 20mM EDTA (pH 8.0) (Shanghai Yu Bo Biotech co., Ltd).
The CTAB extract consisted of: 2% (w/v) CTAB (Shanghai-leaf biotechnology, Ltd), 100mM Tris-HCl (pH 8.0) (Beijing Solarbio), 20mM EDTA (pH 8.0) (Shanghai Yu Bo Biotech co., Ltd), 2.5mol/L NaCl (west longa science, Ltd), 20% PVP40 (Shanghai-leaf biotechnology, Ltd).
And (3) taking the DNA sample, determining the DNA concentration by using a BioSpec-nano micro ultraviolet spectrophotometer, simultaneously recording OD260/OD230 and OD260/OD280, and adjusting the concentration to 50 ng/mu L to obtain a DNA template.
(2) Design of primers
The primer sequence is shown as SEQ ID NO: 1 to 6.
(3) PCR amplification
PCR amplification System: 2 XTaq PCR premix reagent 12.5. mu.L, JQBHS-F primer 0.3. mu.L of 10. mu. mol/L, JQBHS-R primer 0.3. mu.L of 10. mu. mol/L, CLS-F primer 0.3. mu.L of 10. mu. mol/L, CLS-R primer 0.3. mu.L of 10. mu. mol/L, JHS-F primer 0.3. mu.L of 10. mu. mol/L, JHS-R primer 0.3. mu.L of 50 ng/. mu.L of DNA template, and deionized water 9.7. mu.L. And oscillating and uniformly mixing the liquid, and performing instantaneous centrifugation to obtain the PCR amplification system.
PCR amplification procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; finally, extension is carried out for 5min at 72 ℃.
(4) Electrophoretic detection
And adding 5 mu L of 6 × loading buffer into the amplification product, uniformly mixing, spotting 6 mu L of the mixed product on 2% agarose gel, performing electrophoresis for 60min under the condition of 120V voltage, performing Gelred color development, and observing and recording the result in a gel imager.
If the electrophoresis pattern contains a DNA band of 160bp, the sample to be detected contains the Bungarus Parvus medicinal material, the standard Bungarus Parvus decoction or the traditional Chinese medicine formula granule of the Bungarus Parvus; if the map contains a DNA band of 207bp, the sample to be detected contains a red-chain snake medicinal material, a red-chain snake standard decoction or red-chain snake traditional Chinese medicine formula particles; if the map contains 191bp DNA bands, the sample to be detected contains Bungarus fasciatus medicinal material, standard Bungarus fasciatus decoction or Bungarus fasciatus traditional Chinese medicine formula particles.
Example 3 authentication method verification
Samples of Agkistrodon acutus, Elaphe carinata and Bungarus fasciatus (as shown in Table 1) were taken.
The samples were identified using the identification method established in example 2. Wherein, in the PCR system, the amount of the DNA template is 50 ng.
TABLE 1 sample table
The identification result is shown in fig. 1, and it can be seen from the figure that 160bp bands exist in the Bungarus parvus medicinal material, the Bungarus parvus standard decoction (freeze-dried powder), the Bungarus parvus traditional Chinese medicine formula particles, the mixed product containing the Bungarus parvus medicinal material and the mixed product containing the Bungarus parvus standard decoction; the band was not obtained in any of the other snake samples. As can be seen from the figure, the red-chain snake medicinal material, the red-chain snake standard decoction (freeze-dried powder), the mixture containing the red-chain snake medicinal material and the mixture containing the red-chain snake standard decoction both have bands of 207 bp; the band was not obtained for any other snake samples. As can be seen from the figure, the golden cypress medicinal material, the golden cypress standard decoction (freeze-dried powder), the mixture containing the golden cypress medicinal material and the mixture containing the golden cypress standard decoction have a strip of 191 bp; the band was not obtained for any other snake samples. The result shows that the accuracy of the identification method in the invention reaches 100%.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Sequence listing
<110> Guangdong-side pharmaceutical Co., Ltd
<120> primers for multiple PCR identification of Bungarus Parvus medicinal materials, standard decoction and traditional Chinese medicine formula granules, application thereof and identification method
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggagcaatca attttattac aacat 25
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gatcggttaa aagtattgta actgc 25
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
accgtgtacc cgccactg 18
<210> 4
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aagcatgata gcagtaatta gcaca 25
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cctgggtcac ttctaggtag c 21
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gctggtggga gtagtcagaa ac 22
Claims (14)
1. A primer for multiple PCR identification of Bungarus Parvus medicinal materials, standard decoction and traditional Chinese medicine formula granules is characterized by comprising a first primer pair, a second primer pair and a third primer pair;
wherein the sequence of the upstream primer of the first primer pair is shown as SEQ ID NO: 1, and the sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification;
the sequence of the upstream primer of the second primer pair is shown as SEQ ID NO: 3, the sequence of the downstream primer is shown as SEQ ID NO: 4 is shown in the specification;
the sequence of the upstream primer of the third primer pair is shown as SEQ ID NO: 5, the sequence of the downstream primer is shown as SEQ ID NO: and 6.
2. The use of the primer according to claim 1 in (1) or (2):
(1) identifying whether the sample to be detected is bungarus parvus, red-chain snake and/or bungarus multicinctus;
(2) preparing a kit for identifying bungarus parvus, Elaphe carinata and/or Bungarus fasciatus.
3. The use of claim 2, wherein the bungarus parvus is a herbal, standard decoction or a herbal granule;
the red-chain snake is medicinal material, standard decoction or Chinese medicinal formula granule;
the Bungarus fasciatus is prepared from medicinal materials, standard decoction or Chinese medicinal formula granules.
4. The use of the primer according to claim 1 in (1) or (2):
(1) identifying whether the sample to be detected contains bungarus parvus, red-chain snake and/or bungarus multicinctus;
(2) preparing a kit for identifying whether the sample to be detected contains bungarus parvus, red-chain snake and/or bungarus multicinctus.
5. A kit comprising the primer of claim 1.
6. The method for identifying the primers for multiple PCR identification of the Bungarus Parvus medicinal materials, the standard decoction and the traditional Chinese medicine formula granules according to claim 1 is characterized by comprising the following steps:
extracting the genome DNA of a sample to be detected;
performing PCR amplification by using the genomic DNA as a template and the primer as claimed in claim 1, wherein if the amplification product contains a DNA band of 160-170bp, the sample to be detected contains Bungarus Parvus medicinal material, Bungarus Parvus standard decoction and/or Bungarus Parvus traditional Chinese medicine formula particles;
if the amplification product contains a DNA band of 201-210bp, the sample to be detected contains a red-chain snake medicinal material, a red-chain snake standard decoction and/or red-chain snake traditional Chinese medicine formula particles;
if the amplification product contains 190-200bp DNA band, the sample to be detected contains a Bungarus fasciatus medicinal material, standard Bungarus fasciatus decoction and/or Bungarus fasciatus traditional Chinese medicine formula particles.
7. The identification method according to claim 6, wherein the genomic DNA of the sample to be tested is extracted by the following method:
adding CTAB precipitation liquid and proteinase K precipitation into a sample to be detected, and extracting for 2-3 times; adding CTAB extracting solution and beta-mercaptoethanol into the precipitate for extraction, and then adding chloroform-isoamylol for extraction for 2-3 times; and (3) adding isopropanol or isopropanol-sodium acetate into the extracted supernatant for precipitation and extraction, washing and incubating the precipitate, and dissolving the precipitate with water to obtain the genomic DNA of the sample to be detected.
8. The identification method according to claim 6, wherein the genomic DNA of the sample to be tested is extracted by the following method:
taking 0.3-0.8 g of a sample to be detected, grinding the sample to be detected into powder, putting the powder into a centrifuge tube, adding 1-1.8 mL of CTAB precipitation solution and 15-25 mu L of proteinase K, uniformly mixing, heating at 50-60 ℃ for 45-65 min, cooling to room temperature, centrifuging, and removing supernatant; adding 800-1000 mu L CTAB precipitation solution and 15-25 mu L proteinase K, uniformly mixing, heating at 50-60 ℃ for 45-65 min, cooling to room temperature, centrifuging, and removing supernatant; adding 800-1000 mu L of CTAB extracting solution and 5-15 mu L of beta-mercaptoethanol into the precipitate, uniformly mixing, heating at 60-70 ℃ for 100-150 min, cooling to room temperature, taking supernatant, adding chloroform-isoamyl alcohol mixed solution with the same volume, shaking, uniformly mixing, centrifuging, taking 700-800 mu L of supernatant, adding chloroform-isoamyl alcohol mixed solution with the same volume, shaking, uniformly mixing, centrifuging, taking 400-500 mu L of supernatant, adding isopropanol or isopropanol-sodium acetate mixed solution with the same volume, standing at-30 to-20 ℃ for 30-60 min, centrifuging, discarding supernatant, washing the precipitate with ethanol for 2-4 times, discarding supernatant, incubating the precipitate at 35-38 ℃ for 20-40 min, volatilizing ethanol, adding 30-50 mu L of sterilized water for dissolving, and obtaining the genomic DNA of the sample to be detected.
9. The method of claim 7 or 8, wherein the CTAB precipitation solution comprises CTAB, Tris-HCl, EDTA and water; wherein the concentration of CTAB is 1-3% (w/v), the concentration of Tris-HCl is 80-120 mmol/L, and the concentration of EDTA is 10-30 mmol/L;
the CTAB extracting solution comprises CTAB, Tris-HCl, EDTA, NaCl, PVP40 and water; wherein the concentration of CTAB is 1-3% (w/v), the concentration of Tris-HCl is 80-120 mmol/L, and the concentration of EDTA is 10-30 mmol/L; the concentration of NaCl is 1-3 mol/L, and the concentration of PVP40 is 10-30% (w/v).
10. The identification method according to claim 6, wherein the template, the primer, the PCR premix and water are mixed uniformly to obtain a PCR amplification system;
amplifying the PCR amplification system according to a preset amplification program to obtain an amplification product;
carrying out electrophoretic analysis on the amplification product, recording an electrophoretic map of the amplification product, and if the map contains a DNA strip of 160bp, determining that the sample to be detected contains Bungarus Parvus medicinal materials, Bungarus Parvus standard decoction and/or Bungarus Parvus traditional Chinese medicine formula particles;
if the map contains a DNA band of 207bp, the sample to be detected contains a red-chain snake medicinal material, a red-chain snake standard decoction and/or red-chain snake traditional Chinese medicine formula particles;
if the map contains 191bp DNA bands, the sample to be detected contains the Bungarus fasciatus medicinal material, the standard Bungarus fasciatus decoction and/or the Bungarus fasciatus traditional Chinese medicine formula particles.
11. The method of claim 10, wherein the PCR amplification system consists of:
PCR premix 12.5. mu.L; the upstream primer and the downstream primer of the first primer pair are respectively 0.3 mu L, the upstream primer and the downstream primer of the second primer pair are respectively 0.3 mu L, the upstream primer and the downstream primer of the third primer pair are respectively 0.3 mu L, the template is 1 mu L, ddH2O 9.7μL。
12. The identification method of claim 10, wherein the PCR amplification procedure is:
pre-denaturing the amplification system at 92-95 ℃ for 4-6 min, then circulating for 30-40 times under a preset program, and finally extending at 71-75 ℃ for 4-6 min;
wherein the preset program is: the amplification system is denatured at 94-96 ℃ for 28-32 s, annealed at 60-62 ℃ for 26-32 s, and then extended at 70-73 ℃ for 28-35 s.
13. The identification method of claim 12, wherein the PCR amplification procedure is:
pre-denaturing the amplification system at 95 ℃ for 5min, then circulating for 35 times under a preset program, and finally extending at 72 ℃ for 5 min;
wherein the preset program is: the amplification system was denatured at 95 ℃ for 30s, then annealed at 61 ℃ for 30s, and then extended at 72 ℃ for 30 s.
14. The method of claim 10, wherein the electrophoretic analysis method is: and diluting the amplification product, then spotting the diluted amplification product on 1.5-2% agarose gel, performing electrophoresis for 45-60 min under the condition of 115-125V voltage, and performing Gelred color development to obtain an electrophoresis pattern.
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